SUMMARY Quorum sensing is a type of cell-cell communication in bacteria that allows members of a population to coordinate certain behaviors in a cell density-dependent manner. In many species quorum sensing promotes virulence. Quorum sensing involves LuxI-family signal synthases and LuxR-family signal receptors. Cognate LuxI-LuxR pairs are genetically linked, although some LuxRs, called orphans, are not genetically linked to a LuxI. In most described cases the LuxR signals are acyl-homoserine lactones (acyl-HSLs). This research program is focused on an orphan LuxR-family protein, BtaR4, which is required for virulence in the bacterium Burkholderia pseudomallei. BtaR4 is unusual because it responds to an antibiotic, malleilactone, and not acyl- homoserine lactones. The long-term goal is to better understand orphan LuxRs and their role in infections, and evaluate their potential as therapeutic targets. The objective of this application is to characterize the mechanism of BtaR4 in activating virulence genes in vitro and in vivo. The central hypothesis is that BtaR4 is required for virulence because it transcriptionally activates genes required for production malleilactone, which is a key virulence factor, and that activation requires malleilactone itself or host association. This is based on evidence that malleilactone and BtaR4 are required for activation of the malleilactone biosynthetic genes, and malleilactone synthesis is likely also activated by BtaR4 in the host. This proposal aims to i) identify the mechanism responsible for malleilactone-dependent activation of BtaR4, ii) determine the role of BtaR4 in regulating malleilactone production during infections in a C. elegans host and iii) determine how the B. pseudomallei BtaR4 homolog functions using an attenuated BSL-2-approved strain. The contribution of the proposed research is expected to be an understanding of how the QS regulator BtaR4 activates virulence in laboratory culture and in the host. This contribution will be significant because it is expected to provide information to evaluate BtaR4 as a potential target for therapeutic development and expand our current understanding of the basic biology of LuxR-family proteins. Ultimately, such knowledge may lead to new strategies to control quorum sensing and treat or prevent diseases.